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CRISPR mediated live imaging of Genome editing and transcription done for the first time.

Crispr, dubbed as the discovery of the century has been used in variety of applications. It is used in genome editing, epigenetic modification, modulation of gene expression, locus identification (CASFISH). Recently it has also been used in drug delivery. Crispr has revolutionised biology.

Traditional fluorescent in situ hybridization (FISH) requires DNA denaturation, while fluorescent labeled-dCas9 assembled in vitro with sgRNA (CASFISH) detects genomic loci only in fixed samples.  Here, this study report a live imaging approach, CRISPR LiveFISH for the first time, that allows real-time study of various chromosomal functions in living cells. They developed CRISPR LiveFISH, an approach that deploys fluorescent gRNA probes assembled with dCas9 to target and label genomic sequences in living cells. They tested CRISPR LiveFISH on cells derived from patients with Patau Syndrome, a Chr13 trisomy genetic disorder that results in organ defects, intellectual and physical impairment, and is eventually fatal. Here are some of the live cell imaging they have done.
CRISPR LiveFISH tracks dynamics of Chr13 loci in a representative normal amniotic fluid cells. Two Chr13 loci are labeled by Atto565-crRNA (gRNA, red). Hoechst 33342 (blue) labels the cell nucleus. Images were taken with seven Z confocal planes.

CRISPR LiveFISH tracks dynamics of Chr13 loci in a representative PS patient-derived amniotic fluid cell. Three Chr13 loci are labeled by Atto565-crRNA (gRNA, red). Hoechst 33342 (blue) labels the cell nucleus.

CRISPR LiveFISH enables multi-loci chromosome imaging in living primary human T lymphocytes. Simultaneous labeling of Chr13 (Atto488-crRNA, green) and Chr3 (Cy3-crRNA, red) by co-delivery of two CRISPR RNP complexes in living primary human T cells. The recorded T lymphocyte moved slowly to the bottom right, with its movement being slowed down on a collagen pre-treated plate.





Tracking dynamics of translocated endogenous chromosomes in living U2OS cells.Simultaneous labeling of Chr3 (Atto488-gRNA, green) and Chr13 (Atto565- gRNA, red) by co-delivery of labeling gRNAs and cutting gRNAs in complex with Cas9 in living U2OS cells. A translocated chromosome is marked by a pair of closely located Chr3 and Chr13 sites in the center of nucleus.

Tracking formation of translocation between endogenous chromosomes in living U2OS cells. Simultaneous labeling of Chr3 (Atto488-gRNA, green) and Chr13 (Atto565-gRNA, red) by co-delivery of labeling gRNAs and cutting gRNAs in complex with Cas9 in living U2OS cells.




 References:
Science, September 5 2019.

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